The present invention is directed to a novel Afx response element comprising a DNA binding site for the human fork head transcription factor Afx, as well as to its use in the screening for genes.
Diabetes and obesity are global health problems. Diabetes is the leading cause of blindness, renal failure, and lower limb amputations in adults, as well as the major risk factor for cardiovascular disease and stroke. Normal glucose homeostasis requires the finely tuned orchestration of insulin secretion by pancreatic beta-cells in response to subtle changes in blood glucose levels, delicately balanced with secretion of counter-regulatory hormones such as glucagon.
Type 1 diabetes or insulin-dependent diabetes mellitus, IDDM, results from autoimmune destruction of pancreatic beta-cells causing insulin deficiency. Type 2 or NIDDM (non-insulin dependent diabetes mellitus) is characterized by a triad of (1) resistance to insulin action on glucose uptake in peripheral tissues, especially skeletal muscle and adipocytes, (2) impaired insulin action to inhibit hepatic glucose production, and (3) dysregulated insulin secretion (R. A. DeFronzo, (1997); Diabetes Reviews, 5, pp. 177-269). After glucose infusion or ingestion (i.e., in the insulin stimulated state), the liver in type 2 diabetic patients overproduces glucose and the muscle glucose uptake is decreased leading to both hyperinsulinemia and hyperglycemia.
Insulin regulates a wide range of biological processes, including glucose transport, glycogen synthesis, protein synthesis, cell growth, and gene expression. Insulin regulates these processes by altering the concentration of critical proteins or by producing activity-altering modifications of pre-existing enzyme molecules. It is clear that insulin can have both positive and negative effects on the transcription of specific genes (R. M. O""Brien, et al. (1996). Gene regulation in Diabetes Mellitus Lippincott-Raven publishers, Philadelphia. pp. 234-242). The genes regulated by insulin encode proteins that have well-established metabolic connection to insulin, but also secretory proteins/hormones, integral membrane proteins, oncogenes, transcription factors, and structural proteins. Not unexpectedly, this type of regulation of gene expression is seen in the primary tissues associated with the metabolic actions of insulin, namely, liver, muscle, and adipose tissue, but also in tissues not commonly associated with these metabolic effects.
The cis/trans model of trancriptional control can be utilized to understand how insulin regulates gene transcription at the molecular level. The fidelity and frequency of initiation of transcription of eukariotic genes is determined by the interaction of cis-acting DNA elements with trans-acting factors. The specific sequence of the cis-acting element determines which trans-acting factor will bind. Several cis-acting elements thai mediate the effect of insulin on gene transcription have recently been defined. These are referred to as insulin response sequences or elements (IRSs/IREs) (R. M. O""Brien, et al. (1996). Gene regulation in Diabetes Mellitus Lippincott-Raven publishers, Philadelphia. pp. 234-242; G. J. P Kops, et al. (1999). Nature, 398, pp. 630-634; S. Guo et al (1999). J.Biol.Chem. 274, 17184-17192; J. E. Ayala et al. (1999). Diabetes, 48, 1885-1889; and S. K. Durham et al. (1999). Endocrinology, 140, 3140-3146). However, it should be mentioned that to date, there is lack of agreement upon a single insulin response element. Also, that formation of heterodimers between two trans-acting factors can alter their ability to activate transcription, their affinity for DNA or sequence specificity.
One important question in the study of insulin-regulated gene transcription is how a signal passes from the insulin receptor in the plasma membrane through the cytoplasm and the nuclear membrane to a specific trans-acting factor binding to an IRE. Well-characterised signal transduction mechanisms downstream of the insulin receptor involve cascades of kinase/phosphatase reactions, including, among others, the phosphatidylinositol 3-kinase (PI3K) pathway (P. J. Coffer, et al. (1998), Biochem. J. 335, pp. 1-13; S. Paradis, et al. (1998); Genes and Development, 12, pp. 2488-2498; B. B. Kahn (1998); Cell, 92, pp. 593-596). Binding of insulin to its cell surface transmembrane receptor stimulates receptor autophosphorylation and activation of the intrinsic tyrosine kinase activity, which results in phosphorylation of several cytosolic docking proteins called insulin receptor substrates (IRSs). IRSs bind to various effector molecules including the 85 kDa regulatory subunit of P13K. This localizes the 110 kDa catalytic domain of P13K to the plasma membrane. The activated P13K phosphorylates membrane bound phosphoinositides (PtdIns), generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These lipids bind to the pleckstrin homology (PH) domain of protein kinase B (PKB, also known as Akt) leading to its accumulation at the cell membrane. The binding causes a conformational change in PKB that makes it more accessible to phosphorylation, which is necessary for its activation. The kinases, which phosphorylate PKB, are themselves targets for lipid products of P13K and are therefore also localized to the membrane. These kinases are called phosphoinositide-dependent protein kinases (PDK1 and PDK2). Activated PKB dissociates from the membrane and moves to the nucleus and other subcellular compartments.
The Insulin-like Pathway in the Nematode Caenorhabditis elegans 
Recent studies in the nematode Caenorhabditis elegans show that a major target of the Akt/PKB homologues, akt-1 and akt-2, is a transcription factor (S. Paradis, et al. (1998); Genes and Development, 12, pp. 2488-2498). An insulin receptor-like signaling pathway regulates C. elegans metabolism, development, and longevity. This pathway is required for reproductive growth and normal metabolism. Mutations in the insulin receptor homologue daf-2 or in the P13K homologue age-1 cause animals to arrest as dauers, shift metabolism to fat storage, and live longer. This regulation of C. elegans metabolism is similar to the physiological role of mammalian insulin in metabolic regulation. Mutations in the gene daf-16, which encodes a fork head transcription factor that acts downstream of the kinases, suppress the effects of mutations in daf-2 or age-1 (S. Ogg et al. (1997); Nature, 389, pp. 994-999; K. Lin et al. (1997); Science, 278, pp. 1319-1322). The principal role of DAF-2/AGE-1 signaling is thus to antagonize DAF-16. Paradis et al. showed further that inactivation of C. elegans Akt/PKB signaling also causes a dauer constitutive phenotype, and that loss-of-function mutations in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling to repress dauer formation. This indicates that DAF-16 is a negatively regulated downstream target of Akt/PKB signaling. DAF-16 contains four consensus sites for Akt/PKB phosphorylation, which indicate that the kinase exert the negative regulatory effect by directly phosphorylating DAF-16 and altering its transcriptional regulatory function.
Human DAF-16 Homologues
The most closely related proteins, identified so far, to DAF-16 are the human fork head transcription factors Afx, FKHR and FKHRL1. Based on amino acid sequence comparison of their fork head DNA-binding domains, Afx, FKHR, and FKHRL1 share about 60-65% identity with DAF-16 (S. Ogg et al. (1997); Nature, 389, pp. 994-999). Afx shares 83% and 81% identity to the fork head domains of FKHR and FKHRL1, respectively (M. J. Anderson et al. (1998); Genomics, 47, pp.187-199). Although this high homology is confined to the fork head domain, amino acid sequences on either side of this domain show little relatedness. However, there are several amino acid stretches outside the fork head domain that show marked sequence conservation. A N-terminal region of 24 amino acids is 75-83% conserved, and the C-terminal ends of each protein where the transactivation domains are located (J. L. Bennicelli et al. (1995). Oncogene, 11, pp. 119-130 and G. J. P. Kops, et al. (1999). Nature, 398, pp. 630-634 ) contain several stretches of homology. The genes for the human DAF-16 homologues were first identified at chromosomal breakpoints in human tumours (A. Borkhardt et al. (1997); Oncogene, 14, pp. 195-202; W. J Fredericks, et al. (1995); Molecular and Cellular Biology, 15, pp. 1522-1535; M. J. Anderson et al. (1998); Genomics, 47, pp.187-199). These tumours were associated with translocation-generated fusion proteins, Afx/mixed-lineage leukemia (MLL) fusion protein in acute leukemias, and PAX3/FKHR fusion protein in alveolar rhabdomyosarcomas. These fork head proteins contain three PKB phosphorylation sites. It has recently been proposed that Afx is a substrate for PKB (S. R. James et al. Recent Res. Devel. Biochem., 1 (1999), pp. 63-76; and G. J. P Kops, et al. (1999); Nature, 398; pp. 630-634). The phosphorylation of Afx increases after insulin stimulation, and this in terms reduces the activity of the transcription factor. Thus, Afx is negatively regulated by PKB.
A. Brunet, et al. demonstrated in Cell 96 (1999); pp. 857-868, that PKB also regulates the activity of FKHRL1. In the presence of survival factors, such as insulin-like growth factor 1 (IGF1) and neurotrophins, PKB phosphorylates FKHRL1, leading to FKHRL1""s retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation.
It has been shown that Afx can activate insulin response element-driven reporter genes (S. R. James et al. Recent Res. Devel. Biochem., 1 (1999), pp. 63-76; and G. J. P Kops, et al. (1999); Nature, 398; pp. 630-634). However, it has not been shown if these are the optimal response elements, if Afx, FKHR, and FKHRL1 show identical or similar DNA-binding characteristics, or how specific they are with regard to DNA binding.
Given a representative sampling of DNA sequences to which a transcription factor will bind, it is possible to generate a specific profile or model which can be applied to identify DNA sequences to which a transcription factor will bind in vitro. Such a model is useful for the identification of genes with a potential binding site for the transcription factor in the promoter, intergenic sequences, or 3xe2x80x2 regions (the introns and sequences which flank the first and last exons). Subset of the found genes can be created, e.g. based on biological knowledge.
The object of the present invention was to find a response element comprising a DNA binding site for the human fork head transcription factor Afx. In accordance with the present invention, a novel Afx response element comprising the nucleotide sequence AACATGTT is hereby provided, said nucleotide sequence having a binding site for the human fork head transkription factor Afx.
The DNA binding specificity of the fork head protein Afx has in accordance with the present invention been identified. The binding site for Afx is a palindromic sequence, AACATGTT.
The present invention provides the basis for future computer analysis for the identification of genes that are potentially regulated by the transcription factor Afx. The use of this found response element is thus useful in the screening for genes that may be used as diabetes drug targets, as well as in bioinformatic analysis of the human genome. Thus, the present invention provides a subset of genes transcriptionally responsive to insulin, said transcription responsive element being useful in the construction and development of assays which enable and facilitates the analysis of genes interacting with the cytokine receptor signaling pathways (e.g. the insulin receptor). Genes found in such screening may in turn be useful in additional screening methods for compounds modifying the insulin receptor signaling pathway.